This year we have continued to identify novel mAbs in several formats as Fabs, scFvs and eAds against cancer-related proteins. These mAbs were tested for their activity against cancer cells in vitro and used for development of novel approaches for multispecific targeting. We have also characterized drugability of some mAbs including their propensity for aggregation. The major accomplishments are summarized below. 1) Most of the therapeutic antibodies approved for clinical use are full-size IgG1 molecules. The interaction of the IgG1 Fc with the neonatal Fc receptor (FcRn) plays a critical role in maintaining their long half-life. We have hypothesized that isolated Fc domains could be engineered to functionally mimic full-size IgG1 (nanoantibodies) but with decreased (10-fold) size. We demonstrated for the first time the successful generation of a soluble, monomeric CH3 domain (mCH3). In contrast to the wild-type dimeric CH3, the mCH3 exhibited pH-dependent binding to FcRn similar to that of Fc. The binding free energy of mCH3 to FcRn was higher than that of isolated CH2 but lower than that of Fc. Therefore, CH3 may contribute a larger portion of the free energy of binding to FcRn than CH2. A fusion protein of mCH3 with an engineered antibody domain (m36.4) also bound to FcRn in a pH-dependent fashion, and exhibited significantly higher neutralizing activity against HIV-1 than m36.4-Fc fusion proteins. The m36.4-mCH3 fusion protein was monomeric, stable, soluble and expressed at a high level in E. coli. We also found that engineering an additional disulfide bond in mCH3 remarkably increased its thermal stability while the FcRn binding was not affected. These data suggest that mCH3 could not only help in the exploration of the dual mechanisms of the CH3 contribution to Fc functions (dimerization and FcRn interactions) but could also be used for the development of candidate therapeutics with optimized half-life, enhanced tissue penetration, access to sterically restricted binding sites and increased therapeutic efficacy. 2) Isolated human immunoglobulin G (IgG) G CH2 domains are promising scaffolds for novel candidate therapeutics. Unlike other human IgG domains, CH2 is not involved in strong interchain interactions and isolated CH2 is relatively stable. However, isolated single CH2 is prone to aggregation. In native IgG and Fc molecules, the N-terminal residues of CH2 from the two heavy chains interact with each other and form hinge regions. By contrast, the N-terminal residues are highly disordered in isolated CH2. We have hypothesized that removal of the CH2 N-terminal residues may not only increase itsthe stability but also itsthe aggregation resistance of the CH2. To test this hypothesis we constructed a shortened variant of IgG1 CH2 (CH2s) where the first seven residues of the N-terminus were deleted. We found that the thermal stability of CH2s was increased by 5C compared to CH2. Importantly, we demonstrated that CH2s is significantly less prone to aggregation than CH2 as measured by Thioflavin T (ThT) fluorescence, turbidity and light scattering. We also found that the CH2s exhibited pH-dependent binding to a soluble single-chain human neonatal Fc receptor (shFcRn) which was significantly stronger than the very weak shFcRn binding to CH2 s as measured by flow cytometry. Computer modeling suggested a possible mode of CH2 aggregation involving its N-terminal residues. Therefore, deletion of the N-terminal residues could increase drugability of CH2-based therapeutic candidates. This strategy to increase stability and aggregation resistance could also be applicable to other Ig-related proteins to increase their stability and aggregation resistance. 3) Glypican-3 (GPC3) has emerged as a candidate therapeutic target in hepatocellular carcinoma (HCC). However, the oncogenic role of GPC3 in HCC is poorly understood. A novel human engineered antibody domain (HN3) with high avidity (KD = 0.6 nM) for cell surface-associated GPC3 molecules was identified from one of our libraries. HN3 recognized a conformational epitope that requires both the amino and carboxy terminal domains of GPC3. It inhibited proliferation of GPC3-positive cells and exhibited significant HCC xenograft tumor growth inhibition in nude mice. The underlying mechanism of HN3 action may involve cell cycle arrest at G1 phase through yap signaling. This study suggests a novel mechanism for GPC3-targeted cancer therapy. 4) Monoclonal antibodies against mesothelin are being evaluated for the treatment of mesothelioma and multiple forms of cancers, and show great promise for clinical development for solid cancers. Antibodies against mesothelin have been shown to act via immunotoxin-based inhibition of tumor growth and induction of antibody-dependent cellular cytotoxicity (ADCC). However, complement-dependent cytotoxicity (CDC), which is considered one of the most important cell killing mechanisms of therapeutic antibodies against tumors, is inactive for such antibodies. A phage display antibody engineering technology and synthetic peptide screening was used to identify SD1, an engineered human antibody domain to mesothelin. The SD1 human antibody domain recognizes a novel conformation epitope at the C terminal end (residues 539-588) of mesothelin close to the cell surface. To investigate SD1 as a potential therapeutic agent a recombinant human Fc (SD1-hFc) fusion protein with an immunotoxin (SD1-PE38) was generated by fusing SD1 to a truncated form of Pseudomonas exotoxin (PE38). Interestingly, the SD1-hFc protein exhibited strong CDC activity, in addition to ADCC, against mesothelin-expressing tumor cells. The SD1-PE38 immunotoxin inhibited both 2D monolayer and 3D spheroid growth of cancer cells in vitro. Furthermore, the SD1-hFc protein caused significant tumor growth inhibition of tumor xenografts in nude mice. These results demonstrate that the SD1 human antibody has potential as a novel cancer therapeutic candidate, and may significantly improve current antibody therapy targeting mesothelin-expressing tumors. 5) Immune targeting of B-cell malignancies using chimeric antigen receptors (CARs) is a promising new approach, but critical factors impacting CAR efficacy remain unclear. To test the suitability of targeting CD22 on precursor B-cell acute lymphoblastic leukemia (BCP-ALL), lymphoblasts from 111 patients with BCP-ALL were assayed for CD22 expression and all were found to be CD22-positive, with median CD22 expression levels of 3500 sites/cell. Three distinct binding domains targeting CD22 were fused to various TCR signaling domains plus/minus an IgG heavy chain constant domain (CH2CH3) to create a series of vector constructs suitable to delineate optimal CAR configuration. CARs derived from the m971 anti-CD22 mAb, which targets a proximal CD22 epitope demonstrated superior antileukemic activity compared with those incorporating other binding domains, and addition of a 4-1BB signaling domain to CD28.CD3 constructs diminished potency, whereas increasing affinity of the anti-CD22 binding motif, and extending the CD22 binding domain away from the membrane via CH2CH3 had no effect. We conclude that second-generation m971 mAb-derived anti-CD22 CARs are promising novel therapeutics that should be tested in BCP-ALL.